KR101612885B1 - Methods for Detection Methylation on Promoter and Methods for Detection and Tracking Epigenetics Alterations by DNA methylation Using Nanoplasmonic Biosensor - Google Patents

Abstract

The present invention relates to a method for detecting methylation on a promoter using a nanoplasmonic biosensor and a method for detecting and tracking the following genetic mutation by DNA methylation. More particularly, the present invention relates to a method for detecting methylation of a CpG region on a promoter using a nanoplasmonic biosensor As well as a method for evaluating the inhibition of transcription factor steric hindrance and epigenetic mediated transcription due to CpG site methylation, as well as methods for the detection and methylation of CpG.
The methylation analysis method of the promoter using the nanoplasmonic biosensor of the present invention can measure the methylation status and the number of the CpG site immediately without pretreatment such as bisulfate treatment and simultaneously measure the methylation status of the p53 promoter and the overexpressed MBD2 And can be applied and applied to early cancer diagnosis. In addition, since the transcriptional activity change due to the methylation of the promoter can be measured, the transcriptional activity according to the epigenetic mediator can be tracked and evaluated.

Description

[0001] The present invention relates to a method for detecting methylation on a promoter using a nanoplasmonic biosensor, and a method for detecting and tracking the epigenetic mutation by DNA methylation,

The present invention relates to a method for detecting methylation on a promoter using a nanoplasmonic biosensor and a method for detecting and tracking the following genetic mutation by DNA methylation. More particularly, the present invention relates to a method for detecting methylation of a CpG region on a promoter using a nanoplasmonic biosensor As well as a method for evaluating the inhibition of transcription factor steric hindrance and epigenetic mediated transcription due to CpG site methylation, as well as methods for the detection and methylation of CpG.

Numerous diseases are caused by gene abnormalities, and the most frequent form of the gene is a change in the genetic code sequence. These gene changes are called mutations. When a mutation is present in a gene, the structure and function of the protein encoded by the gene change, an abnormality and cleavage occur, and the mutated protein causes disease. However, even if there is no mutation in a specific gene, an abnormality may occur in expression of the gene, which may cause disease.

A typical example is methylation in which a methyl group is attached to a cytosine base site in a gene transcription regulatory region, for example, a CpG island. This is referred to as epigenetic change, and it exhibits the same effect as transition to progeny cells in a manner similar to mutation, for example, loss of expression of the corresponding protein. In the case of cancer cells, the expression of the tumor suppressor gene is inhibited by the methylation of the promoter CpG island, and this suppressed expression is recognized as one of the important mechanisms causing cancer (Robertson, K. and Jones, P., Carcinogen, 21, 461, 2000). In fact, abnormal methylation / demethylation of CpG islands has been reported in various cancer cells such as prostate cancer, colon cancer, uterine cancer and breast cancer, and it has been shown that they play an important role in the early stage of cancer development. It is attracting attention as a marker.

Thus, the promoter methylation of tumor-associated genes is an important indicator of cancer, which can be used in many ways, including diagnosis and early diagnosis of cancer, prediction of carcinogenesis risk, prediction of cancer prognosis, have. Recently, an attempt has been made to use the methylation specific PCR (hereinafter referred to as MSP) or an automatic base analysis method for the diagnosis and screening of cancer. Recently, there has been a vigorous research in the field of blood, sputum, saliva, stool, (M. Estet, M. et < RTI ID = 0.0 > t < / RTI > al ., Cancer Res ., 59: 67,1999; Sanchez-Cespedez, M. et al ., Cancer Res ., 60: 892,2000; Ahlquist, da et al ., Gastroenterol . , 119: 1219,2000).

In particular, the promoter of p53, which is known as a tumor suppressor gene, has a transcription factor (TF) binding site (AP1, NF-kB, YY1, c-Myc), which contains a low density of CpG sequences (A. Ventura, meat al ., Nature , 445: 661, 2007; CC Harris and M. Hollstein, N Engl J Med ., 329: 1318, 1993). When the CpG site at the low density is irregularly methylated (methylated CpG; mCpG), MBD2 (Methyl-CpG-binding domain protein 2) binds to induce transcriptional repression (W. Dai et al ., Clin Cancer Res. , 15: 5788, 2013; A. Chatagnon, et al ., Epigenetic , 11: 1295, 2011). At the transcriptional regulatory level, minimal mutation regulatory methylation plays an important role in inactivating the transcription of the p53 gene, MBD2 is an important transcription repressor and a potential tumor biomarker, and A / T adjacent to CpG in vivo and ex vivo Regardless of the sequence, it is known that efficient binding to a single mCpG or all mCpG moieties on the p53 promoter (dsp53) and binding of MBD2 results in steric hindrance and inhibition of transcription (YM Omar et < RTI ID = 0.0 > al ., Mol Cancer Res , 9: 1152, 2011; E. Ballestar et al ., The EMBO J. 23: 6335, 2003). In addition, if both the methylated p53 promoter (mdsp53) and overexpressed MBD2 are present in the actual sample, the biological phenomenon associated with the onset of tumorigenesis is observed, so that both the p53 promoter and MBD2 can be applied to the diagnostic test with the biomarker.

However, despite the recent advances in methylation detection methods, it is possible to simultaneously detect the methylated p53 promoter and MBD2 in a sample, to observe the steric hindrance of the transcription factor by MBD2, and to track or evaluate the suppression of epigenetic mediated transcription Development of multi-detection platform is required.

Recently, biosensor technology has focused on the development of non-labeling measurement technology that does not use labeling material. Typically, the method using surface plasmon resonance (RQ DUAN et al ., Neoplasma , 59 (3): 348, 2012; Wei Zhou meat al ., International Journal of Nanomedicine , 6: 381, 2011), a method for measuring the refractive index change of light due to the bending phenomenon of a three-dimensional microstructure, cantilever (Balle, MK. Et al, Ultramicroscopy, 82;. 1 , 2000), method of measuring the modified crystal resonant frequency change in accordance with the mass change of the biomolecule (Marx, KA, Biomacromolecules, 4 ;. 1099, 2003), based on the semiconductor process How to measure the electric field effect (Yuqing, M. et al ., Biotechnol . Adv . , 21; 527, 2003) and an electrochemical measurement method (Hanahan, G. et al . , J. Environ . Monit . , 6; 657, 2004; Sang Hee Han et al., Analytica Chimica Acta , 665: 79, 2010).

Since the localized surface plasmon resonance (LSPR) method can quantitatively detect the reaction without complicated pre-purification and labeling processes, many studies on the interaction between biomolecules immobilized on the surface Has been used. In the LSPR method, when light having various wavelengths is irradiated to a material having a localized surface such as metal nanoparticles, polarization is generated on the surface of the metal nanoparticles, unlike bulk metals, and the characteristic of electric field is remarkable. These LSPR optical properties are sensitive to changes in the permittivity (refractive index) near the nanoparticles. This change in dielectric constant (refractive index) can be used to detect adsorption between biomolecules. Over the last decades, the optical properties of LSPR have been based on the biomaterial concentration, thickness, and binding reaction rate data for specific biochemical analytes including antigen / antibody, ligand / receptor, protein / protein reaction and DNA hybridization Various biomolecule interaction analyzes have been performed.

Therefore, in the present invention, as a result of intensive efforts to develop a novel multiple detection platform capable of simultaneously detecting the methylated p53 promoter and MBD2 and tracking the suppression of epigenetic mediated transcription, gold nanostar (AuNS) and PNA Using a nanoplasmonic biosensor manufactured using a probe, it is possible to simultaneously and rapidly detect methylated p53 promoter and MBD2 in real time without labeling, It was confirmed that the transcriptional activity can be measured, and the present invention was completed.

It is an object of the present invention to provide a method for detecting a methylated CpG region of a target nucleic acid and a method for quantifying mCpG using a nanoplasmonic biosensor.

The present invention also provides a method for detecting transcription activity by detecting the presence or absence of methylation in the transcription factor binding domain on the promoter and suppressing transcriptional genetic mediated transcription by promoter methylation.

(A) a PNA probe hybridizing with the 5 'end or 3' end portion of the target nucleic acid is vertically or horizontally immobilized, or a PNA probe hybridizing with a portion of the target nucleic acid is aligned horizontally Treating the immobilized gold nanostar-based plasmonic sensor with a target nucleic acid to form a gold nanostar-PNA probe-target nucleic acid complex; (b) contacting the complex formed with methylated CpG-specific protein to induce binding to the CpG site of the target nucleic acid; And (c) analyzing the light scattering spectrum resulting from binding of the methylated CpG-specific protein and analyzing whether or not the target nucleic acid CpG region is methylated through the maximum wavelength mobility (? Max) analysis obtained therefrom. A method for detecting methylation of a CpG region.

(A) a PNA probe hybridizing to the 5'end or 3'end of the target nucleic acid is immobilized vertically or horizontally, or a PNA probe hybridizing to a portion of the target nucleic acid is immobilized horizontally, (B) contacting the formed complex with a MBD2 (Methyl-CpG-binding domain protein 2) protein to contact the starch-based plasmonic sensor with a target nucleic acid to form a gold nanostar-PNA probe-target nucleic acid complex; Gt; CpG < / RTI > site of the target nucleic acid; And (c) measuring the light scattering spectrum due to the MBD2 protein binding of the methylated CpG site and measuring the number of methylated CpG sites of the target nucleic acid through the maximum wavelength mobility analysis obtained therefrom. Lt; RTI ID = 0.0 > CpG < / RTI >

(A) a PNA probe hybridizing to the 5'end or 3'end of the target nucleic acid is immobilized vertically or horizontally, or a PNA probe hybridizing to a portion of the target nucleic acid is immobilized horizontally, Treating a star-based plasmonic sensor with a sample containing a target nucleic acid to form a gold nanostar-PNA probe-target nucleic acid complex; (b) contacting MBP2 (Methyl-CpG-binding domain protein 2) protein to the complex thus formed to induce binding to the methylated CpG site of the target nucleic acid; And (c) analyzing the light scattering spectrum according to MBD2 protein binding of the methylated CpG region and analyzing whether the CpG region of the target nucleic acid is methylated through the maximum wavelength mobility (? Max) analysis obtained therefrom. The present invention provides a method for providing information necessary for the diagnosis of cancer caused by methylation.

The present invention also provides a method for producing a PNA probe, comprising the steps of: (a) preparing a gold nanostart in which a PNA probe hybridizing with the 5 'terminal or 3' terminal portion of the promoter is fixed vertically or horizontally, or a PNA probe hybridizing with the center portion of the promoter is horizontally fixed Treating a plasmonic sensor based on a promoter to form a gold nanostar-PNA probe-promoter complex; (b) contacting the formed complex with MBD2 (Methyl-CpG-binding domain protein 2) protein to induce binding to the methylated CpG site of the transcription factor binding domain, and then adding a transcription factor; And (c) a step of measuring the light scattering spectrum due to the binding of the transcription factor, and detecting whether the CpG region is methylated in the transcription factor binding domain in the promoter through analysis of the maximum wavelength mobility and the reaction rate constant obtained therefrom Lt; RTI ID = 0.0 > CpG < / RTI > region of the transcription factor binding domain.

The present invention also provides a method for producing a PNA probe, comprising the steps of: (a) preparing a gold nanostart in which a PNA probe hybridizing with the 5 'terminal or 3' terminal portion of the promoter is fixed vertically or horizontally, or a PNA probe hybridizing with the center portion of the promoter is horizontally fixed Treating a plasmonic sensor based on a promoter to form a gold nanostar-PNA probe-promoter complex; (b) contacting the formed complex with a methylated CpG-binding domain protein 2 (MBD2) protein to induce binding to the methylated CpG site of the target nucleic acid, and then treating the RNA polymerase and the transcription factor; And (c) analyzing the activity of the promoter by analyzing the maximum wavelength mobility and the reaction rate constant obtained from the measurement of the light scattering spectrum due to the binding of the RNA polymerase and the transcription factor, and comparing the activity There is provided a method for measuring inhibition of transcriptional activity by methylation of a CpG region of a promoter comprising the step of analyzing transcription activity.

The present invention also provides a method for producing a PNA probe, comprising the steps of: (a) preparing a gold nanostart in which a PNA probe hybridizing with the 5 'terminal or 3' terminal portion of the promoter is fixed vertically or horizontally, or a PNA probe hybridizing with the center portion of the promoter is horizontally fixed Treating a plasmonic sensor based on a promoter to form a gold nanostar-PNA probe-promoter complex; (b) contacting the formed complex with RNA polymerase, MBD2 and a transcription factor to induce binding to the CpG region of the promoter; (c) The light scattering spectrum according to the binding of MBD2, RNA polymerase and transcription factor is measured, and the competitive interaction of the transcription inhibitor and the transcription factor protein is monitored through analysis of the maximum wavelength mobility and the reaction rate constant obtained therefrom A method for monitoring the competitive interaction of a transcription factor protein with methylation of a promoter CpG region comprising the steps of:

The methylation analysis method of the promoter using the nanoplasmonic biosensor of the present invention can measure the methylation status and the number of the CpG site immediately without pretreatment such as bisulfate treatment and simultaneously measure the methylation status of the p53 promoter and the overexpressed MBD2 And can be applied and applied to early cancer diagnosis. In addition, since the transcriptional activity change due to the methylation of the promoter can be measured, the transcriptional activity according to the epigenetic mediator can be tracked and evaluated.

1 is a schematic diagram for detecting multiple targets using the nanoplasmonic biosensor of the present invention.
Fig. 2 is a data showing the UV / VIS pattern (A) and the size and shape (B) of the gold nanostar produced in the present invention.
FIG. 3 is a graph showing the degree of hybridization according to the pH of the PNA probe and the p53 promoter according to pH.
4 is data showing the PNA probe specificity of the nanoplasmonic biosensor of the present invention and mCpG binding ability of MBD2 protein.
FIG. 5 is a schematic view of a PNA probe bonded vertically or horizontally to a gold nanostar surface. FIG.
6 is data obtained by observing the degree of hybridization by infrared spectroscopy (FT-IR; A) and electrophoresis (B) when the PNA probe is immobilized on a gold nanostar surface vertically or horizontally.
FIG. 7 is a graph showing the wavelength shift (A) according to the methylated p53 promoter and MBD2 binding, the mCpG number in the p53 promoter (B), and the maximum wavelength shift (C and B) according to the MBD2 treatment concentration.
8 is a schematic diagram (A) for a sensitive region of the plasmon resonance region around the gold nanostar and a schematic diagram (B) for horizontal coupling for increasing the stability of the PNA probe fixation.
Fig. 9 shows data obtained by fixing the mRNA on the gold nanostar surface with the PNA probe vertical (A) or horizontal (B) and confirming the position of mCpG in the p53 promoter.
FIG. 10 shows the results of confirming total DNA methylation degree (A) and MBD2 (B) in HeLa cells and HEK293 cell nuclear extracts.
Fig. 11 is a graph showing the wavelength shifts when the p53 promoter of the HeLa cell (A) and the HEK293 cell (B) and the positive control group (mdsp53; C) were bound to the gold nanostars immobilized with the PNA probe, and when the MBD2 was bound to mCpG It is the data which observes the degree of wavelength shift.
Fig. 12 shows the results of simultaneous detection of methylation of the p53 promoter of HeLa cells and HEK293 cells, MBD2 of each cell (A) and correlation of the maximum wavelength shift according to the number of mCpG and mCpG in the p53 promoter of HeLa cells and HEK293 cells And the relationship (B).
13 is data showing transcription factor steric hindrance due to binding of MBD2 to mCpG.
14 is a schematic diagram of a method for measuring transcription activity or transcriptional repression using a nanoplasmonic biosensor. In the plasmon-inactive region 2, an RNA polymerase is ligated to a plasmon sensitive region 1, To measure the spectral change that occurs as it moves to
FIG. 15 shows the inhibition of epigenetic mediated transcription according to the methylated promoter. When the MBD2-containing nuclear extract of HeLa cells was treated with (A) MBD2, the maximum wavelength mobility of (B) (C and D) of the observed data and the methylated transcription factor binding domain p53 promoter (mAP-1 dsp53, mNF-kB dsp53, mYY1 dsp53).
Fig. 16 is a data obtained by observing the transcriptional repression inhibitory activity upon MBD2 addition through the analysis of maximum wavelength mobility after the initiation of transcription.
FIG. 17 shows the inhibition of posterior archaeal mediated transcription by methylation of the respective transcription factor binding domains. It was found that when MBD2 was treated (A) and when MBD2 was not treated (B), the maximum wavelength mobility to be.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention detects a methylated promoter and a target nucleic acid by using a nanoplasmonic biosensor. Since many CpG sites on the genome are actually methylated, it is possible to specifically recognize the sequence and detect methylation of a target nucleic acid or a specific promoter We used a capture probe to immobilize the PNA probe to gold nanoparticles. PNA (Peptide Nucleic Acid) is a nucleic acid analogue having a basic skeleton of N- (2-aminoethyl) glycinamide. Since the PNA skeleton is electrically neutral, it has higher specificity than a DNA probe for a target nucleic acid having a complementary base sequence (Nuclease) or protease, and is widely used in molecular diagnostic methods using probes (Egholm et < RTI ID = 0.0 > al ., Nature , 365: 556,1993; Nielsen et al ., Bioconjugate Chem ., 5: 3, 1994; Demidov, et al., Biochem . Pharmacol ., 48: 1310,1994).

In the present invention, a PNA probe was synthesized so that three hydrocarbon (γ) -binding positions were included in a hydrocarbon chain including a (C ₃ -NH ₂ ) linker at the amino group end of PNA, and a gamma (γ) modification resulted in increased promoter-specific complementarity and facilitation of PNA-DNA complex formation.

In the present invention, gold nanostar (AuNS) was synthesized using PNA as an integrated platform with localized surface plasmon resonance (LSPR) system, and LSPR was used for the change of optical properties of gold nanostar It is very sensitive, and thus it is possible to detect, in a specific, simple, real-time manner, the epigenetic change caused by the methylation of the promoter. Based on Rayleigh's theory, local surface plasmon resonance-based nanoplasmonic biosensors can be used to measure the degree of methylation and inhibition of progeny-mediated transcription by analyzing the refractive index and spectral wavelength shifts occurring in the vicinity of gold nanostars One).

In one aspect, the present invention relates to a method for detecting a nucleic acid comprising (a) a PNA probe hybridizing with a 5 'end or 3' end portion of a target nucleic acid is fixed vertically or horizontally, or a PNA probe hybridizing with a portion of the target nucleic acid is horizontally fixed Treating a gold nanostar-based plasmonic sensor with a target nucleic acid to form a gold nanostar-PNA probe-target nucleic acid complex; (b) contacting the complex formed with methylated CpG-specific protein to induce binding to the CpG site of the target nucleic acid; And (c) analyzing the light scattering spectrum resulting from binding of the methylated CpG-specific protein and analyzing whether or not the target nucleic acid CpG region is methylated through the maximum wavelength mobility (? Max) analysis obtained therefrom. And a method for detecting methylation of a CpG region.

In another aspect, the present invention provides a method for detecting a nucleic acid molecule comprising: (a) a PNA probe hybridizing with a 5'end or 3'end of a target nucleic acid is vertically or horizontally immobilized, or a PNA probe hybridizing with a portion of the target nucleic acid is horizontally immobilized (B) a step of treating a target nucleic acid with a gold nanostar-based plasmonic sensor to form a gold nanostar-PNA probe-target nucleic acid complex; (b) adding a methylated CpG-binding domain protein 2 To induce binding to the methylated CpG site of the target nucleic acid; And (c) measuring the light scattering spectrum due to the MBD2 protein binding of the methylated CpG site and measuring the number of methylated CpG sites of the target nucleic acid through the maximum wavelength mobility analysis obtained therefrom. And a method for measuring the number of CpG sites.

In another aspect, the present invention provides a nucleic acid probe comprising: (a) a PNA probe that hybridizes with a 5 'end or 3' end portion of a target nucleic acid is vertically or horizontally immobilized, or a PNA probe that hybridizes with a portion of the target nucleic acid is horizontally immobilized Treating a gold nanostar-based plasmonic sensor with a sample containing a target nucleic acid to form a gold nanostar-PNA probe-target nucleic acid complex; (b) contacting MBP2 (Methyl-CpG-binding domain protein 2) protein to the complex thus formed to induce binding to the methylated CpG site of the target nucleic acid; And (c) analyzing the light scattering spectrum according to MBD2 protein binding of the methylated CpG region and analyzing whether the CpG region of the target nucleic acid is methylated through the maximum wavelength mobility (? Max) analysis obtained therefrom. The present invention relates to a method for providing information necessary for the diagnosis of cancer caused by methylation of cancer cells.

(A) a PNA probe hybridizing with the 5 'end or the 3' end portion of the promoter is vertically or horizontally fixed, or a PNA probe hybridizing with the center portion of the promoter is horizontally immobilized Forming a gold nanostar-PNA probe-promoter complex by treating a nanostar-based plasmonic sensor with a promoter; (b) contacting the formed complex with MBD2 (Methyl-CpG-binding domain protein 2) protein to induce binding to the methylated CpG site of the transcription factor binding domain, and then adding a transcription factor; And (c) a step of measuring the light scattering spectrum due to the binding of the transcription factor, and detecting whether the CpG region is methylated in the transcription factor binding domain in the promoter through analysis of the maximum wavelength mobility and the reaction rate constant obtained therefrom RTI ID = 0.0 > CpG < / RTI >

(A) a PNA probe hybridizing with the 5 'end or the 3' end portion of the promoter is vertically or horizontally fixed, or a PNA probe hybridizing with the center portion of the promoter is horizontally immobilized Forming a gold nanostar-PNA probe-promoter complex by treating a nanostar-based plasmonic sensor with a promoter; (b) contacting the formed complex with a methylated CpG-binding domain protein 2 (MBD2) protein to induce binding to the methylated CpG site of the target nucleic acid, and then treating the RNA polymerase and the transcription factor; And (c) analyzing the activity of the promoter by analyzing the maximum wavelength mobility and the reaction rate constant obtained from the measurement of the light scattering spectrum due to the binding of the RNA polymerase and the transcription factor, and comparing the activity To a method for measuring the inhibition of transcriptional activity by methylation of a CpG region in a promoter comprising the step of assaying transcriptional activity.

(A) a PNA probe hybridizing with the 5 'end or the 3' end portion of the promoter is vertically or horizontally fixed, or a PNA probe hybridizing with the center portion of the promoter is horizontally immobilized Forming a gold nanostar-PNA probe-promoter complex by treating a nanostar-based plasmonic sensor with a promoter; (b) contacting the formed complex with RNA polymerase, MBD2 and a transcription factor to induce binding to the CpG region of the promoter; (c) The light scattering spectrum according to the binding of MBD2, RNA polymerase and transcription factor is measured, and the competitive interaction of the transcription inhibitor and the transcription factor protein is monitored through analysis of the maximum wavelength mobility and the reaction rate constant obtained therefrom The present invention relates to a method for monitoring the competitive interaction of a transcription factor protein with methylation of a promoter CpG region comprising the steps of:

In the present invention, the target nucleic acid and the promoter are p53 promoters. However, the present invention is not limited thereto, and various promoters and target nucleic acids can be used.

In the present invention, the PNA probe is 5 'amine terminus (NH ₂₎ group, or are combined, and characterized in that the amine (NH ₂₎ group is bonded to the inner PNA probe, the amine group (NH ₂₎ groups PNA probe And is coupled to the gamma position of the skeleton.

When the amine (NH ₂ ) group is bonded to the 5 'end, the PNA probe is fixed to the gold nanostructure vertically. When the amine (NH ₂ ) group is bonded to the PNA probe, the PNA probe is bound to the gold nanostructure And is fixed horizontally.

In the present invention, the gold nanostar-based plasmonic sensor in step (a) may include (i) fixing a star-shaped gold nanostar to a transparent support selected from glass or plastic; (CH ₂ ) ₁₁ (OCH ₂ CH ₂ ) ₆ OCH ₂ COOH was immobilized on a gold nanostar fixed surface, and then a PNA probe specific for target nucleic acid (CH ₂ ) was detected using EDC / NHS, ₁₁ (OCH ₂ CH ₂ ) ₆ OCH ₂ COOH.

In order to fabricate a nanoplasmonic biosensor based on the surface plasmon resonance phenomenon of the present invention, star-shaped gold nanostars having a size of about 45 nm were prepared by a known method (surfactant-stabilized seed; TK Sau et al . , Adv . Mater, 22: 1805, and attached to 2010) were synthesized in (2), fixed on a glass substrate and a glass substrate, wherein the imaging chamber gold nanoparticles are fixed (RC-30, Warner Instrument Inc.), the imaging The chamber was inserted into a sample holder of a dark field resonance microscope to produce a nanoplasmonic sensor. Subsequently, HS (CH ₂ ) ₁₁ (OCH ₂ CH ₂ ) ₆ OCH ₂ COOH (OEG _{6 -} COOH) was attached to the surface of the gold nanostar, and a PNA probe having a sequence specific to the target nucleic acid or probe was dissolved in EDC / NHS bioconjugation, and bound to OEG _{6 -} COOH. The target nucleic acid, probe, and MBD2 protein were sequentially bound to measure the maximum wavelength shift through the change of the refractive index around the gold nanostar.

In the present invention, the complex formation of the PNA probe and the target nucleic acid in the step (a) is performed at a reaction temperature of 50 to 55 ° C and a pH of 5.4 to 8.3.

As a result of confirming the hybridization conditions of p53 promoter and PNA in the present invention, it was confirmed that optimal hybridization was observed at 52 ° C and pH 5.4 (FIG. 3). Analysis of the specificity of pNA promoter and specificity for mCpG binding , The maximum wavelength shift of DNA 1, DNA 2 and DNA 3, which are nonspecific sequences in the PNA probe, was negligible while the maximum shift value of the DNA was significantly increased when the p53 promoter was treated. In addition, it was confirmed that the maximum wavelength shift value was changed only when MBD2 was injected into the nanoplasmonic sensor, and it was confirmed that mCpG of the p53 promoter was specifically recognized and bound (FIG. 4).

In the present invention, the measurement of the light scattering spectrum in the step (c) is performed using a dark-field microscope and a Rayleigh scattering spectrum, The mechanical resonance noise signal of the field resonance microscope itself was measured and spectrum analysis was performed. The LSPR scattering maximum wavelength shift value (λ _max ) obtained from the change of the refractive index of the single gold nanoparticle was calculated by the following equation .

[Equation 1]

Δλ _max = lambda _max (after binding) -? _max (before binding)

In the present invention, the treatment concentration of the MBD2 protein is in the range of 120 to 130 fM. In order to confirm the treatment concentration of the optimal MBD2, treatment is performed at a concentration of 5 x 10 ^-6 fM to 125 x 10 ⁶ fM, As a result, it was confirmed that the maximum wavelength shift value was increased in a concentration dependent manner, and it was confirmed that LSPR λ _max shift of 16.3 nm was observed at 125 fM, thereby showing a remarkable sensitivity (FIGS. 7C and 7D).

As shown in FIG. 5, the present invention can be used by immobilizing a PNA probe vertically or horizontally on a gold nanostar surface according to the purpose of detection. The formation of the PNA-dsp53 promoter complex on gold nanostars when the PNA probe was coupled vertically or horizontally was analyzed using Fourier transform infrared spectroscopy (FT-IR) (FIG. 6A) and electrophoresis (FIG. 6B) .

In the present invention, when the PNA probe was immobilized on the gold nanostar surface vertically and the methylated p53 promoter and MBD2 were injected, the maximum wavelength shifted to the right for each immobilization step, and PNA And mdp53 were hybridized, and when MBD2 was bound to mCpG, the plasmonic wavelength shifted toward the red wavelength at 33.8 nm and 49.5 nm, respectively, as compared with gold nanostar (Fig. 7A).

To determine the degree of LSPR maximum wavelength shift and the degree of saturation of LSPR maximal wavelength shift depending on the number of mCpG present in the promoter (target nucleic acid), target DNA was synthesized so that there were 1 to 4 mCpG on the DNA sequence, As a result of measuring the LSPR maximum wavelength mobility according to the number of mCpG present in the target nucleic acid, the change of the maximum wavelength shift according to the number of methylated CpG was confirmed, and the maximum wavelength shift value was increased depending on the methylated CpG When the methylated CpG sites were 3 and 4, the maximum wavelength shift values were 24 nm and 24.8 nm, respectively, and the degree of saturation of the LSPR spectral shift was confirmed (FIG. 7B).

When the PNA probe is vertically bonded to the gold nanostar, the wavelength shift according to the change in length can be analyzed to measure the number of mCpG, the presence of methylation, the methylation position, the steric hindrance and the transcription activity. The length of the target DNA (target nucleic acid) , The analysis can be performed by horizontally bonding the PNA probe to the gold nanostar. The gold nanostar used in the present invention has a region corresponding to about 20 nm from the surface as a plasmon sensitive region. Since each of the sequences containing each mCpG has a length of 30 bp, about 10.3 nm (10 bp About 3.4 nm) and is included in the sensitive region of the surface plasmon resonance region (Fig. 8A). However, when the length of the target DNA becomes longer, it becomes difficult to judge the presence or absence of methylated mCpG in the region outside the sensitive region. When the PNA probes are horizontally bonded to gold nanostars, the position of methylated mCpG from the gold nanostars is the same, so that the number of mCpG and the steric hindrance due to MBD2 can be measured.

In addition, a PNA probe sequence specific to the p53 promoter was synthesized, immobilized vertically on gold nanostars, and then p53 promoter was introduced into the chamber to induce hybridization with the PNA probe. However, Resulting in the separation of PNA probes from gold nanostars. In order to stably immobilize the PNA probes on gold nanostars, PNA probes were prepared by introducing -NH ₂ groups (marked with * in the above sequence) at two sites of gamma (?) Binding sites in the PNA sequence. To still bind perpendicularly to the gold nanostar surface, a PNA probe was made to bind to the 5 ' end of the p53 promoter (Fig. 8B).

As a result of measuring the LSPR maximum wavelength mobility according to the position of mCpG present in the pro-motor (target nucleic acid) when the PNA probe was immobilized on gold nanostars vertically or horizontally, When fixed, it was confirmed that the maximum wavelength shift value was 16.5 nm, 15.2 nm, 13.8 nm and 13.2 nm, respectively, depending on the positions of -105, -77, -35 and +1 on the p53 promoter (FIG. 9A). On the other hand, when the PNA probe is immobilized on the gold nanostar surface horizontally, the hybridization of the horizontally fixed PNA probe and the p53 promoter is not influenced by the mCpG position of the p53 promoter because the length from the gold nanostar is the same regardless of the mCpG position It was confirmed that the maximum wavelength shifts were similar to each other (FIG. 9B).

In the present invention, an experiment was conducted to confirm whether the methylated p53 promoter and MBD2 can be multiplexed using a nanoplasmonic biosensor. In this case, MBD2 performs both a target substance and an mCpG detector do.

Experiments were performed by separating the p53 promoter fragment and the nuclear extract containing MBD2 from HEK 293 cells (Hk; CRL-1573 ^TM ) and HeLa cells (He; CCL-2 ™) as a sample. As a result, The p53 promoter (He-dsp53) showed a wavelength shift similar to that of the positive control (mdsp53), but the p53 promoter of HEK293 cells showed minimal migration of the wavelength (Fig. 11). This means that there is a high level of methylated CpG in the p53 promoter of HeLa cells, which is similar to the degree of total methylation in Fig. 10A of Example 4-1. In the case of HEK293 cells, methylated CpG in the p53 promoter is almost present It means not to do.

In the simultaneous detection of the degree of methylation of p53 promoter and MBD2, when MBD2 was reacted with the p53 promoter of HeLa cells as shown in Fig. 12A, recombinant MBD2 (Rec MBD2), He-MBD2 and Hk- nm, 16.4 nm, and 2.5 nm, indicating that MBD2 was overexpressed in HeLa cells, which was confirmed to be similar to the MBD2 quantitation value of FIG. 10B of Example 4-1. When the MBD2 was reacted with the p53 promoter of HEK293 cells, there was almost no methylated portion in the p53 promoter of HEK293 cells, and it was confirmed that the wavelength change due to MBD2 treatment was not large.

Further, in order to monitor the steric hindrance of the transcription factor due to the methylation of the electromagnetic factor binding domain on the p53 promoter, each basic promoter active region (-213 to +1) and short RNA-encoding DNA fragment (+1 to +30 ) Were synthesized and used in the experiments. MBD2 protein was treated on the nanoplasmonic biosensor, and then the transcription factor of each of AP-1, NF-κB, YY-1 and Myc And RNA polymerase II (p33 subunit). When MBD2 was bound, the maximum wavelength shift of 16.3 nm was measured. However, when the transcription factor was reacted, it was observed that there was no further migration to the red wavelength (FIG. 13). This suggests that MBD2 is completely bound to mCpG of the transcription factor binding domain and that important transcription factors are not coupled due to steric hindrance.

To measure the transfer rate, a PNA probe was vertically immobilized on a gold nanostar to measure the LSPR spectrum, which is dependent on RNAP II, which moves closer to the gold nanostar surface. When the PNA probe is vertically bound to the gold nanostar, mCpG is located outside of the plasmonic sensitive region (Fig. 14). When RNAP II binds from the outside of the promoter and moves along the p53 promoter, Changes occur in real time. If MBD2 in the nuclear extract binds to mCpG on the p53 promoter, the change in LSPR is less significant than that of the p53 promoter in which the LSPR changes are not methylated, so that changes in the progeny genetic mediated transcriptional activity can be measured.

In the present invention, the binding rate of RNA polymerase (RNAP II) was measured on the methylated promoter in the presence of MBD2 in order to measure the transcription rate upon methylation of the p53 promoter. As shown in Table 5 and Fig. 14, in the case of the methylated p53 promoter, formation of a pre-initiation complex was inhibited in MBD2 binding, and the methylation-free p53 promoter The change in the maximum wavelength shift of the LSPR was not large (Figs. 15A and 15B). The wavelength change depending on the presence of MBD2 is judged as a difference due to MBD2 binding.

The decrease in the maximum LSPR shift value of the LSPR was confirmed by MBD2, which is slower than that of the hk-dsp53 and umdsp53 promoters in mdsp53 and he-dsp53.

The relative transcriptional activity of the p53 promoter and the methylated transcription factor binding domain p53 promoter (mAP-1 dsp53, mNF-kB dsp53, mYY1 dsp53) from HeLa cells and HEK cells was measured using a nanoplasmonic biosensor, Were reduced to 43%, 35% and 27% in mAP-1, mNF-kB and mYY1, respectively. He-dsp53, hk-dsp53 and mdsp53 were reduced to 45%, 86% and 29% (Fig. 15C and D).

In order to confirm whether the transcription was initiated by MBD2, each transcription factor (AP-1, NF-κB, YY-1, c-Myc) and RNA polymerase was introduced into the chamber of the nanoplasmonic sensor After the induction of the transcription reaction, MBD2 was injected into the chamber, and the inhibition of transcription by MBD2 was observed. As a result, it was confirmed that when the mCpG was present in the transcription factor binding domain, the maximum wavelength shift value decreased (Fig. 16), indicating that MBD2 binds to mCpG of the transcription factor binding domain, Can be.

No methylated p53 promoter (SEQ ID NO: 6), methylated p53 promoter (positive control, SEQ ID NO: 5) was used to measure the degree of suppression of epigenetic mediated transcription by the presence of methylated CpG in each transcription factor binding domain. (SEQ ID NOS: 23 and 25, Table 6, respectively), which contained mCpG and methylated mCpG. As a result, the presence of MBD2 bound to mCpG in the transcription factor binding domain (Mdsp53) and methylated mCpG under AP-1 and NF-κB transcription factor binding domains were reduced compared to unmethylated p53 promoter (umdsp53) (FIG. 17) .

In conclusion, in the present invention, it was confirmed that methylation determination and quantification, simultaneous detection of mCpG and overexpressed MBD2 protein on the promoter, and suppressive activity of progeny genetic mediated transcription can be measured using a nanoplasmonic biosensor It can be confirmed that the transcriptional repression inhibitor of the cancer suppressor gene such as p53, which is a biological feature in the early stage of cancer development, is due to transcriptional repression due to methylation in the transcription factor binding domain on the promoter.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Nanoplasmic  Biosensor Fabrication

1-1: Gold Nanostar  Produce

In order to fabricate a nanoplasmonic biosensor based on the surface plasmon resonance phenomenon of the present invention, star-shaped gold nanostars having a size of about 45 nm were prepared by a known method (surfactant-stabilized seed; TK Sau et al., Adv . Mater, 22: 1805, was synthesized as 2010), it was purchased from the jaeryoeun Sigma Aldrich (Sigma Aldrich, USA) used in the synthesis.

The gold nanostart seed solution was prepared by mixing 0.5 ml of 10 mM HAuCl ₄ and 15 ml of 0.1 M CTAB and 1.2 ml of sodium borohydride (NaBH ₄ , a reducing agent) in a cold state to grow gold nanostructured seeds Was added to and dissolved in the light brown seed solution (seed solution). The solution for gold nanostar growth was prepared by mixing 9.5 ml of 0.1 M CTAB, 0.06 ml of 10 mM AgNO ₃ and 0.4 ml of 10 mM HAuCl ₄ (growth solution), adding HAuCl ₄ Change. To start the growth of the gold nanostar, 20 μl of the seed solution was added to the growth solution and 0.064 ml of ascorbic acid 100 mM (the solution turned to a clear color). Then 0.05 ml of 0.1 M NaOH was added and the gold nanostar was grown by stirring at 250 rpm on a magnetic stirrer for about 2 hours and 30 minutes until the color of the solution changed from purple / blue to dark green.

When the growth of the gold nanostar was completed, the solution was filtered with a 0.2 mu l filter to remove the agglomerated product from the solution in which the reaction was completed, to obtain a uniform size of gold nanostar for the production of the nanoplatemonic biosensor (HR-TEM, jEOL JEM-3011) and UV / VIS (high resolution transmission electron microscopy) were used for the analysis. The samples were centrifuged at 26,000 rpm for 1 hour using 20-80% sucrose density purchased centrifugation. Using a spectrophotometer (UV / VIS 3600, Shimadzu).

As a result, it was confirmed that a gold nanostar having a size of about 45 nm was fabricated, and it was confirmed that a maximum wavelength value was obtained at about 700 nm (FIG. 2).

1-2: Gold Nanostar  Fixed and chamber  setting( chamber setting )

The gold nanostars prepared in Example 1-1 were fixed on a coverslip slide (22 x 40 x 0.1 mm) activated with 3-MPTES.

First, the cover slip slide was ultrasonicated in a piranha solution (H ₂ SO ₄ : H ₂ O ₂ : 3: 1) and then thoroughly washed with 18.2 mΩ / m ultra pure water. The solution containing gold nanostars was diluted to an OD 0.02 value at 690 nm absorbance and then reacted for 30 seconds to electrostatically fix the glass substrate (cover slide glass) to the center of the drop-coating. Here, the concentration and the reaction time of the gold nanoparticles serve to reduce the inter-particle coupling effect by controlling the intergranular spacing and the density of the fixed gold nanostars.

Thereafter, the prepared organic substrate sample was mounted on a microfluidic imaging chamber (RC-30, Warner Instrument Inc.), and the imaging chamber was inserted into a sample holder of a dark field resonance microscope (Eclip TE2000-U, Nikon, Japan) . The imaging chamber is connected to a syringe pump, which is a means for injecting various chemicals such as adsorbates and reactants necessary for surface functionalization of gold nanoparticles, and the flow rate of the fluid flowing into the chamber is adjusted to 50 μl / min. The optimum bonding temperature in the chamber was controlled using a thermostat (TC-324B, Warner Instruments).

After system setting, the fixed gold nanostar surface was washed with absolute ethanol for 5 minutes, then ultra-pure distilled water was drained for 10 minutes to remove unattached gold nanostars and other impurities. Then, 1 ml of 0.2 mM HS (CH ₂ ) ₁₁ (OCH ₂ CH ₂ ) ₆ OCH ₂ COOH (OEG _{6 -} COOH) contained in ethanol was injected into the chamber and reacted for 10 hours to attach to the gold nanostar surface .

The PNA probes were then ligated to the OEG ₆ COOH (SEQ ID NO: 2) through an EDC / NHS bioconjugation reaction using an NHS coupling kit (Thermo Scientific, FL, USA) to immobilize a PNA probe specific for the target promoter or target nucleic acid ≪ / RTI >

Nanoplasmic  Biosensor PNA  And p53  Promoter Hybridization  Identify conditions and specificity

2-1: Hybridization  Condition check

In order to understand the degree of hybridization between the PNA probe and the p53 promoter and whether methylation was detected in the nanoplasmonic biosensor prepared in Example 1, a portion of the basic activity promoter region (BAP) of about 213 bp of the p53 promoter in the human genomic DNA, The full-activity promoter (381 bp) was subjected to PCR using the following two primer sets (bioneer synthesis).

Primer set Base sequence SEQ ID NO: P53Bac-F 5'-GGGAGAAAACGTTAGGGTGTGG-3 ' SEQ ID NO: 1 P53Bac-R 5'-CCAATCCAGGGAAGCGTGTCAC-3 ' SEQ ID NO: 2 P53P-F 5'-TGCACCCTCCTCCCCAACTC-3 ' SEQ ID NO: 3 P53P-R 5'-CTCCCTGGACGGTGGCTCTAG-3 ' SEQ ID NO: 4

The BAP portion of the p53 promoter was reacted at 94 ° C for 4 minutes, followed by 30 cycles of 94 ° C for 30 seconds, 57 ° C for 30 seconds, and 72 ° C for 1 minute, and the entire active promoter portion was amplified at 94 ° C After 4 minutes of reaction, the reaction was repeated 30 times at 94 ° C for 30 seconds, 56 ° C for 30 seconds, and 72 ° C for 1 minute, and the products were separated and used in subsequent experiments by methods known in the art.

In order to determine the hybridization conditions with the PNA probe and the p53 promoter (dsp53) fixed to the gold nano-star, a PNA and dsp53 concentration of 3: 1 to ensure that the perfectHyb ^TM plus hybridization buffer (1 ×; Sigma Aldrich) at 52 ° C. The pH of the reaction was 5.4, 6.2, 7.0, 7.7, and 8.3, respectively. In order to observe the hybridization efficiency, 2.5% agarose gel electrophoresis was performed, and the result was analyzed using a UV transilluminater (Spectronics, NY, USA). As a result, it was confirmed that optimal hybridization was observed at pH 5.4 (FIG. 3) .

2-2: Identification of specificity

In order to confirm the specificity of the nanoplasmonic biosensor, the specificity of the PNA probe to the p53 promoter and the specificity to the mCpG binding were analyzed.

In order to confirm the specificity of the PNA probe, a PNA sequence specific to the p53 promoter was synthesized, immobilized on the gold nanostructure of the nanoplasmonic sensor prepared in Example 1, the nonspecific DNA and p53 promoter were respectively treated, The maximum wavelength shift value was analyzed by the change analysis. The p53 promoter was treated with methyltransferase (CpG Methyltranferase; M.SssI, New England Biolabs, USA) in the p53 promoter so that the CpG portion was methylated on the promoter (used as a positive control in the subsequent experiments).

Light scattering spectra were measured using a dark-field resonance microscope at each stage of immobilization. For the processing of data, the Lorentzian algorithm of the OriginPro 7.5 program was applied to the spectrum obtained as a result of the experiment, Respectively. Finally, the LSPR scattering maximum wavelength shift (λ _max ) obtained from the change in the refractive index of the single gold nanoparticle was calculated by the following equation (1).

[Equation 1]

Δλ _max = λ _max (after binding) -λ _max (before binding)

The mdsp53 promoter sequence and the PNA sequence name Base sequence SEQ ID NO: Mdsp53 promoter 5'-CAGGT mCGGmCG AGAATCCTGACTCTGCACCCTCCTCCCCAACTCCATTTCCTTTGCTTCCTC mCG GCAGG mCG GATTACTTGCCCTTACTTGTCATGG mCG ACTGTCCAGCTTTGTGCCAGGAGCCT mCG CAGGGGTTGATGGGATTGGGGTTTTCCCCTCCCATGTGCTCAAGACTGG mCG CTAAAAgttttgagcttctcaaaagtctagagccacc-3 ' SEQ ID NO: 5 umsp53 promoter 5'-CAGGTCGGCGAGAATCCTGACTCTGCACCCTCCTCCCCAACTCCATTTCCTTTGCTTCCTCCGGCAGGCGGATTACTTGCCCTTACTTGTCATGGCGACTGTCCAGCTTTGTGCCAGGAGCCTCGCAGGGGTTGATGGGATTGGGGTTTTCCCCTCCCATGTGCTCAAGACTGGCGCTAAAAgttttgagcttctcaaaagtctagagccacc-3 ' SEQ ID NO: 6 PNA for Mdsp53 promoter 5'-NH2 linker-AATCCGCCTGCCGGA-3 ' SEQ ID NO: 7 DNA-1 (oligo from human GAPDH gene) (NCBI JN613429) 5'-TGCAGCAATGCCTCCTGCACCACCAACTGCTTAG-3 ' SEQ ID NO: 8 DNA-2 (oligo from elongator complex gene 3) (NCBI) 5'-GACAAATACACACACAGCCTTGCCGTGAATTG-3 ' SEQ ID NO: 9 DNA-3 (Oligo from SP6 promoter) 5'-GGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAAT-3 ' SEQ ID NO: 10

In addition, after hybridization of the p53 promoter and the p53 promoter containing the mCpG portion, human IgG, MeCP2, P53 protein and MBD2 protein (New England Biolabs, USA) were individually treated to confirm binding specifically to mCpG.

As a result, it was confirmed that the maximum wavelength shift of DNA 1, DNA 2 and DNA 3, which are non-specific sequences in the PNA probe, was minimal, while the maximum shift value of DNA was rapidly increased when the p53 promoter was treated. In addition, it was confirmed that the maximum wavelength shift value was changed only when MBD2 was injected into the nanoplasmonic sensor, and it was confirmed that mCpG of the p53 promoter was specifically recognized and bound (FIG. 4).

MeCP2 is known to recognize mCpG as well as MBD2, but MeCP2 is not suitable for detection of mCpG on the p53 promoter because it is influenced by the presence of A / T sequence around mCpG.

PNA Probe  And methylated p53  Changes in wavelength due to promoter binding and MBD2  Confirm protein concentration

3-1: Depending on the vertical or horizontal direction PNA Probe  Confirm immobilization degree

In the present invention, a PNA probe was vertically or horizontally immobilized on a gold nanostar surface according to the purpose of detection (FIG. 5). In the immobilization of the PNA probe, -COOH of OEG _{6 -} COOH immobilized on the gold nanostar surface reacts with -NH ₂ present at the 5 'end of the PNA probe, and the PNA probe is horizontally (Using a C ₃ -NH ₂ linker) to modify the position of -NH ₂ in the PNA probe sequence to two positions.

In order to analyze the fixing efficiency of the PNA fixed vertically or horizontally on the gold nanostar surface, the PNA probe corresponding to SEQ ID NO: 6 of Example 2 was used. In the case of vertically binding PNA probes, the -NH ₂ group was introduced only at the 5 'end, and in the case of the horizontally binding PNA probe, the -NH ₂ group at two sites of gamma (γ) bonding in the PNA sequence as well as the 5' (Indicated by * in the sequence) was introduced to prepare a PNA probe.

When the EDC / NHS coupling agent was treated with a gold nanostar surface functionalized with OEG ₆ COOH using Fourier transform infrared spectroscopy (FT-IR), it was confirmed that PNA was immobilized (FIG. 6A) The formation of the PNA-dsp53 promoter complex on the star was confirmed by electrophoresis (Fig. 6B).

3-2: Gold Nanostar  Vertical to surface Combined PNA The probe  When used, methylated p53  Promoter and MBD2  Confirmation of wavelength shift due to coupling

PNA probes were immobilized on the gold nanostar surface perpendicularly to the nanoplasmonic biosensor prepared in Example 1, and the changes in wavelength when the methylated p53 promoter and MBD2 were injected were measured.

The methylated p53 promoter (mdsp53) and the PNA probe used the sequence in Table 2, and the PNA probe was bound to OEG ₆ COOH on the gold nanostar surface in the same manner as in Example 1. The mdsp53 promoter was introduced into the chamber and reacted at 52 캜, pH 5.4 for 2 hours. To prevent nonspecific binding, the temperature was raised to 64 캜 and the sodium chloride concentration was treated to 100 mM. Thereafter, MBD2 (New England Biolabs, USA) was injected into the chamber after washing with ionized water three times to remove the unattached mdsp53 promoter, followed by incubation at 37 [deg.] C and pH 7.4 to bind the mCpG portion of the p53 promoter Lt; / RTI >

Light scattering spectra were measured using a dark field resonance microscope at each step of immobilization, and the LSPR scattering maximum wavelength shift (λ _max ) obtained from the change in the refractive index of a single gold nanoparticle was calculated.

As a result, it was confirmed that the maximum wavelength shifts to the right for each immobilization step. When the hybridization of PNA and mdsp53 and MBD2 to mCpG were combined, the plasmonic wavelength was 33.8 nm and 49.5 nm, respectively, (Fig. 7A).

In order to confirm the treatment concentration of the optimal MBD2, the maximum wavelength shift value was examined by treating the concentration of 5 × 10 ^-6 fM to 125 × 10 ⁶ fM. As a result, it was confirmed that the maximum wavelength shift value increased in a concentration- LSPR lambda _max shift of < RTI ID = 0.0 > 16.3 < / RTI > nm was observed at 125 fM and was found to exhibit significant sensitivity (Figs. 7C and 7D).

3-3: Measurement of the wavelength change according to the number of methylations and the number of methylation dependent LSPR Degree of saturation  Confirm

In order to determine the degree of LSPR maximum wavelength shift and the degree of saturation of LSPR maximum wavelength shift according to the number of mCpG present in the promoter (target nucleic acid), 1 to 4 mCpGs were present on the DNA sequence. And corresponding PNA sequences were synthesized (Biona, Korea).

mCpG < / RTI > number and a PNA probe mCpG Count Base sequence SEQ ID NO: 1 mCpG 5'-CTTTCGTTCCTC mCG GCAGGCGGATCGCTAA-3 '
SEQ ID NO: 11 2 mCpG 5'-CTTTCGTTCCTC mCG GCAGG mCG GATCCGTAA-3 '
SEQ ID NO: 12 3 mCpG 5'-CTTT mCG TTCCTC mCG GCAGG mCG GATCCGTAA-3 '
SEQ ID NO: 13 4 mCpG 5'-CTTT mCG TTCCTC mCG GCAGG mCG GATC mCG TAA-3
SEQ ID NO: 14 PNA probe _{5'-NH 2 -linker-GAGGAACGAAAG-} 3 '
SEQ ID NO: 15

The gold nanostar used in the present invention has a region corresponding to about 20 nm from the surface as a plasmon sensitive region. Since each of the sequences containing each mCpG has a length of 30 bp, about 10.3 nm (10 bp About 3.4 nm) and is included in the sensitive region of the surface plasmon resonance region (Fig. 8A).

As a result of confirming the change of the maximum wavelength shift value according to the number of methylated CpG, it was confirmed that the maximum wavelength shift value was increased depending on the methylated CpG, and when there were 3 and 4 methylated CpG sites, Were 24 nm and 24.8 nm, respectively, and the degree of saturation of the LSPR spectral shift was confirmed (Fig. 7B).

3-4: Gold Nanostar  Surface vertical or horizontal Combined PNA In the probe  In the promoter mCpG  Location verification

In order to measure the LSPR maximum wavelength mobility according to the mCpG position in the promoter (target nucleic acid), from the transcription start position (+1) at the four mCpG positions in the 213-BAP region of the p53 promoter, Sequences were prepared so that mCpG was included at positions -105, -77, -35, and +1, respectively (Table 4).

Sequence according to mCpG position SEQ ID NO: 1 mCpG site at -105 position 5'-TCCGGCAGGCGGATTACT mCGC CCTTACTTGTCATGGCGACTGTCCAGCTTTGTGCCAGGAGCCTCGCAGGGGTTGATGGGATTGGGGTTTTCCCCTCCCATGTGCTCAAGACTGGCGCTAAAA-3 '(+ 1) SEQ ID NO: 16 1 mCpG site at -77 position 5'-TCCGGCAGGCGGATTACTCGCCCTTACTTGTCATGGCGACTGTCCA mCG TTTGTGCCAGGAGCCTCGCAGGGGTTGATGGGATTGGGGTTTTCCCCTCCCATGTGCTCAAGACTGGCGCTAAAA-3 '(+ 1) SEQ ID NO: 17 1 site at -35 position 5'-TCCGGCAGGCGGATTACTCGCCCTTACTTGTCATGGCGACTGTCCACGTTTGTGCCAGGAGCCTCGCAGGGGTTGATGGGATTGGGGT mCG TCCCCTCCCATGTGCTCAAGACTGGCGCTAAAA-3 '(+1) SEQ ID NO: 18 1 site at +1 position 5'-TCCGGCAGGCGGATTACTCGCCCTTACTTGTCATGGCGACTGTCCACGTTTGTGCCAGGAGCCTCGCAGGGGTTGATGGGATTGGGGTTTTCCCCTCCCATGTGCTCAAGACTGG mCG CTAAAA-3 '(+ 1) SEQ ID NO: 19 PNA probe (vertical) 5'-AATC (*) CGCCTGC (*) CGGA-3 ' SEQ ID NO: 20 PNA probe (horizontal) 5'-GGTCGA (*) AACACGGT (*) CCTC-3 ' SEQ ID NO: 21

(* Above is the portion into which the -NH ₂ group is introduced in the PNA probe sequence)

In addition, a PNA probe sequence specific to the p53 promoter was synthesized, immobilized vertically on gold nanostars, and then p53 promoter was introduced into the chamber to induce hybridization with the PNA probe. However, Resulting in the separation of PNA probes from gold nanostars. In order to stably immobilize the PNA probes on gold nanostars, PNA probes were prepared by introducing -NH ₂ groups (marked with * in the above sequence) at two sites of gamma (?) Binding sites in the PNA sequence. To still bind perpendicularly to the gold nanostar surface, a PNA probe was made to bind to the 5 ' end of the p53 promoter (Fig. 8B).

In the case of a horizontally binding PNA probe, it was synthesized to bind to the middle portion of the p53 promoter, and a -NH ₂ group (indicated by * in the above sequence) was introduced at two sites in the PNA sequence to prepare a PNA probe.

Then, the PNA probe was injected into the nanoplasmonic biosensor prepared in Example 1 to be fixed vertically or horizontally on the surface of the gold nanostar. Then, the -105, -77, -35, and +1 positions Were injected into each well to form a gold nanostar-PNA probe-p53 promoter complex. MBD2 was then treated to induce binding to mCpG on the p53 promoter and light scattering spectra were measured using a dark field resonance microscope to calculate the LSPR scatter maximum wavelength shift (λ _max ).

As a result, when the PNA probe was immobilized on the gold nanostar surface vertically, the maximum wavelength shift value was found to be 16.5 nm, 15.2 nm, 13.8 nm, 13.2 nm according to the -105, -77, -35 and +1 positions on the p53 promoter, nm (Fig. 9A). In other words, when the length of 10 bp was calculated to be 3.4 nm and the position of the mCpG-MBD2-bonded portion was calculated, it was confirmed that the positions were 6.1 nm, 14.5 nm, 28.8 nm and 35.7 nm from the gold nanostar, It was confirmed that the difference of the maximum wavelength shift occurs depending on the position of mCpG.

On the other hand, when the PNA probe is fixed horizontally on the gold nanostar surface, the maximum wavelength shift value is about 16.5 nm, and the maximum wavelength shift according to the mCpG position change is similar (FIG. 9B). This is because hybridization of the horizontally fixed PNA probe and the p53 promoter is not influenced by the mCpG position of the p53 promoter because the length from the gold nanostar is the same regardless of the mCpG position.

Nanoplasmic  Using methylated biosensor p53  Promoter and MBD2  Multiple detection

4-1: methylated p53  Promoter and MBD2  Preparation of materials for multiple detection

Experiments were conducted using HEK 293 cells (Hk; CRL-1573 ™) and HeLa cells (He; CCL-2 ™) to determine the degree of methylation of the p53 promoter and overexpression of MBD2 in actual cell lines.

First, HeLa cell (He; CCL- 2 ™) and HEK-293 cell (Hk; CRL -1573 ™) in the genomic DNA is AccuPrep ^R Genomic DNA Extract Kit (Bioneer, Korea) was used to isolate based on the manual. (TCTAGA, 427/431; New England Biolabs, USA) and BamHI (GGATCC, 73/77; New England Biolabs, USA) were used to obtain 358 bp containing the BAP region of the p53 promoter and a positive control In Example 2-2, a sequence in which all CpGs were methylated using CpG methyltransferase (M.SssI; NEB biolabs, MA, USA) was used.

MBD2 protein of each cell used nuclear extracts of commercially available recombinant MBD2 protein (NEB, USA), HEK 293 cells (Hk; CRL-1573 ™) and HeLa cells (He; CCL-2 ™).

When the degree of methylation of the p53 promoter and the expression level of MBD2 in each cell measured by the nanoplasmonic biosensor of the present invention were measured, the degree of methylation of HeLa cells and HEK 293 cells And the concentration of MBD2 contained in the nuclear extract were measured.

To determine the total degree of methylation of HeLa cells and HEK 293 cells used in the experiments, the assay was performed using Imprint ^® Methylated DNA Quantitation kit (Sigma Aldrich, USA). As a result, (Fig. 10A). In addition, the MBD2 protein contained in the nuclear extracts of HeLa cells and HEK 293 cells was measured by sandwich ELISA using an antibody specific for MBD2 protein. As a result, it was confirmed that MBD2 protein was expressed at a high level in HeLa cells ( 10B)

4-2: methylated p53  Promoter and MBD2  Multiple detection

In order to stably bind the PNA probe to the gold nanostar surface, a PNA probe capable of recognizing the p53 promoter of the HEK 293 cell was constructed, and the -NH ₂ group at two sites in the PNA sequence inside the PNA probe *) Were introduced.

[SEQ ID NO: 22]

PNA probe: 5'-CTGAGA (*) GCAAACGC (*) AAAA-3

(The part marked with * in the sequence is the part where the -NH ₂ group is introduced)

After the PNA probe (SEQ ID NO: 22) was immobilized to stably bind the PNA probe to the gold nanostar surface fixed on the glass substrate in the closed chamber, the p53 promoter fragment isolated in Example 4-1 was inserted into the chamber Injected 52 Deg.] C, pH 5.4 for 2 hours, then treated with 100 mM sodium chloride and the temperature was increased to 64 [deg.] C to remove the nonspecific reaction. As a positive control, the p53 promoter obtained by methylation of all CpG in Table 2 was used.

Then, nuclear extracts of HeLa cells and HEK 293 cells isolated in Example 4-1 and 125fM of MBD2 protein (positive control and mCpG detector; NEB, USA) were injected into the chamber, respectively, and incubated at 37 ° C for 2 hours And the light scattering spectrum using a dark field resonance microscope was measured to calculate the LSPR scattering maximum wavelength shift value (? _Max ).

When the p53 promoter of HeLa cells and HEK 293 cells were bound to the PNA probe and the changes in the spectra when MBD2 was bound to mCpG in the p53 promoter were observed under a dark-field microscope, the HeLa cell p53 promoter (He-dsp53 ) Was observed to have a wavelength shift similar to that of the positive control (mdsp53), but it was confirmed that the p53 promoter of HEK293 cell had a small wavelength shift (Fig. 11). This means that there is a high level of methylated CpG in the p53 promoter of HeLa cells, which is similar to the degree of total methylation in Fig. 10A of Example 4-1. In the case of HEK293 cells, methylated CpG in the p53 promoter is almost present It means not to do.

In the simultaneous detection of the degree of methylation of p53 promoter and MBD2, when MBD2 was reacted with HeLa cell p53 promoter as shown in Fig. 12A, recombinant MBD2 (Rec MBD2), He-MBD2 and Hk-MBD2 showed 21.5 nm, 17.0 nm, and 2.9 nm, indicating that MBD2 was overexpressed in HeLa cells, which was confirmed to be similar to the MBD2 quantitation value of FIG. 10B of Example 4-1.

When the respective MBD2 was reacted with the p53 promoter of HEK293 cells, it was confirmed that the maximum wavelength shift values of recombinant MBD2 (Rec MBD2), He-MBD2 and Hk-MBD2 were 3.0 nm, 2.0 nm and 2.1 nm, This is because there is almost no methylated portion in the p53 promoter of HEK293 cells, and the wavelength change due to MBD2 treatment is not large.

4-3: HeLa  Cells and HEK293  Cell p53  Within the promoter mCpG  Number measurement

In the experiment for measuring the mCpG number in the p53 promoter of HeLa cells and HEK293 cells, it is impossible to measure the mCpG number accurately since methylation does not occur during the DNA replication process when cells of the cell growing period are used. Therefore, The p53 promoter was isolated by the method of Example 4-1 in stasis, and the degree of methylation was different in each cell. Therefore, the p53 promoter was isolated from each of 15 samples.

Further, in order to accurately measure the number of mCpG present in the p53 promoter of HeLa cells and HEK 293 cells, a gold nanostar immobilized on a glass substrate in a closed chamber was immersed in a PNA probe into which -NH ₂ group was introduced at two sites in the PNA sequence (SEQ ID NO: 22).

Then, the nuclear extracts of HeLa cells and HEK 293 cells and 125fM of MBD2 protein (positive control and mCpG detector; NEB, USA) were injected into the chamber, respectively, followed by reaction at 37 ° C for 2 hours, Light scattering spectra were used to calculate the LSPR scattering maximum wavelength shift value (? _Max ).

As a result, approximately mCpG could be measured in the p53 promoter of HeLa cells, and in the case of HEK293 cells, it was confirmed that methylation was not performed in the p53 promoter except for two samples (Fig. 12B).

p53  The electron-binding domain on the promoter ( transcrption factor binding  domain) of the transcription factor by methylation of the transcription factor monitoring

 The methylated transcription factor binding domain sequence containing each of the basic promoter active regions (-213 to +1) and the short RNA-encoding DNA fragment (+1 to +30) is a PNA probe (SEQ ID NO: 20) was immobilized on a cover glass by reacting with gold nanostar fixed horizontally. 125 fM recombinant MBD2 was injected into the chamber, reacted for 1 hour, and washed with 0.5X PBS buffer to remove unbound MBD2. (AP1S2; Mybiosource, USA), NF-κB (p50; Sigma Aldrich, USA), YY-1 (P01; Abnova, Taiwan), c-Myc (SRP 2089-5UG; Sigma Aldrich, USA) The transcription factor and RNA polymerase II (p33 subunit; Sigma Aldrich, USA) were injected into the reaction chamber and reacted for 1 hour.

The rate of transcription factor binding on the methylated transcription factor binding domain to which MBD2 was coupled measured the maximum wavelength shift of the LSPR spectrum.

As a result, it was observed that the maximum wavelength of 16.3 nm was shifted when MBD2 was bound, but it was observed that when the transcription factor was reacted, there was no further migration to the red wavelength (FIG. 13). This suggests that MBD2 is completely bound to mCpG of the transcription factor binding domain and that important transcription factors are not coupled due to steric hindrance.

p53  Measurement of transcription rate by methylation of promoter

In the present invention, the binding rate of RNA polymerase (RNAP II) was measured on the methylated promoter in the presence of MBD2 in order to measure the transcription rate upon methylation of the p53 promoter.

First, the mdsp53 promoter containing exons 1 (+1 to +30) and the unmethylated dsp53 promoter isolated from clinical samples were hybridized with PNA-immobilized gold nanostars. MBD2 is then injected into the chamber, and 37 (AP-1, NF-κB, YY-1, c-Myc) and ribonucleotides were added to induce a transcription reaction.

As shown in Table 5 and Fig. 14, in the case of the methylated p53 promoter, formation of a pre-initiation complex was inhibited in MBD2 binding, and the methylation-free p53 promoter The change in the maximum wavelength shift of the LSPR was not large (Figs. 15A and 15B). The wavelength change depending on the presence of MBD2 is judged as a difference due to MBD2 binding.

The transcriptional activity analysis was analyzed by Min Sun Song et al., Adv . Mater ., 25: 1265, 2013). The reaction rate constant was measured by using the _{k = ln 2 / t 1/} 2 ( equation 2),

In order to analyze the activity of the promoter using the above reaction rate, the expression v = k _t K, which expresses the activity of the promoter, was analyzed by changing to the following Equation 3 due to the following assumptions.

&Quot; (3) "

At this time, k _t K is the binding constant, RV is the relative promoter activity, k _b The association rate constant of the RNA polymerase for the promoter raised, Δλ _max Represents the wavelength shift due to binding of the protein, and the subscript i represents the type of each mutation promoter. Since the wavelength shift value of the spectrum is proportional to the degree of binding of the protein, it can be a measure of the binding affinity, that is, the binding constant.

Transfer speed comparison according to existence of MBD2 Presence of MBD2 Without MBD2 mdsp53 0.547 s ^-1 0.066 s ^-1 he-dsp53 0.227 s ^-1 0.072 s ^-1 mAP1 0.121 s ^-1 0.068 s ^-1 mNF-κB 0.101 s ^-1 0.072 s ^-1 hk-dsp53 0.092 s ^-1 0.035 s ^-1 umdsp53 0.0224 s ^-1 0.0254 s ^-1

The relative transcriptional activity of the p53 promoter and the methylated transcription factor binding domain p53 promoter (mAP-1 dsp53, mNF-kB dsp53, mYY1 dsp53) from HeLa cells and HEK cells was measured using a nanoplasmonic biosensor, Were reduced to 43%, 35% and 27% in mAP-1, mNF-kB and mYY1, respectively. He-dsp53, hk-dsp53 and mdsp53 were reduced to 45%, 86% and 29% (Fig. 15C and D).

In order to confirm whether the transcription was initiated by MBD2, each transcription factor (AP-1, NF-κB, YY-1, c-Myc) and RNA polymerase was introduced into the chamber of the nanoplasmonic sensor After the induction of the transcription reaction, MBD2 was injected into the chamber, and the inhibition of transcription by MBD2 was observed. As a result, it was confirmed that when the mCpG was present in the transcription factor binding domain, the maximum wavelength shift value decreased (Fig. 16), indicating that MBD2 binds to mCpG of the transcription factor binding domain, Can be.

Following methylation of each transcription factor binding domain Posthumous relational  Mediated transcription inhibition measurement

No methylated p53 promoter (SEQ ID NO: 6), methylated p53 promoter (positive control, SEQ ID NO: 5) was used to measure the degree of suppression of epigenetic mediated transcription by the presence of methylated CpG in each transcription factor binding domain. And NF-κB transcription factor binding domain sequences (SEQ ID NOS: 23 and 25, Table 6, respectively), including the methylated mCpG.

AP-1, an NF-KB transcription factor binding domain sequence and a PNA probe name Base sequence SEQ ID NO: NF-kB 5'-GCCAGGAGCCT mCG CAGGGGTTGATGGGTTGGGGTTTTCCCCTCCCATGTGCTCCTGGCGCTAAAAgttttgagcttctcaaa agtctagagccacc-3 SEQ ID NO: 23 NF-kB PNA probe 5'-GTCCCCA (*) ACTACC (*) CTA -3 SEQ ID NO: 24  AP-1 5'-TATCTACGGCACCAGGT mCG G mCG AGAATCCTGACTCTGCACCCTCCTCCCTGGCGCTAAAAgttttgagcttctcaaa agtctagagccacc-3 ' SEQ ID NO: 25 AP-1 PNA probe 5'-CTTAG (*) GACTGAGA (*) CG -3 ' SEQ ID NO: 26

(* Above is the portion into which the -NH ₂ group is introduced in the PNA probe sequence)

PNA probes specific for each target DNA were immobilized on a gold nanostructure horizontally. Then, the target DNA was injected into the chamber and reacted at 52 ° C, pH 5.4 for 2 hours. To prevent non-specific binding, the temperature Lt; RTI ID = 0.0 > 64 C < / RTI > MBD2 is then injected into the chamber, and 37 Lt; 0 > C for 3 minutes, and then each transcription factor and ribonucleotide was added to induce a transcription reaction.

As a result of measurement of spectrum change, AP-1 containing the methylated p53 promoter (mdsp53) and methylated mCpG in the presence of MBD2 bound to mCpG in the transcription factor binding domain, p53 without NF-κB transcription factor binding domain It was confirmed that the transcription rate was decreased as compared with the promoter (umdsp53) (Fig. 17).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Korea University Research and Business Foundation <120> Methods for Detection Methylation on Promoter and Methods for          Detection and Tracking Epigenetics Alterations by DNA methylation          Using Nanoplasmonic Biosensor <130> P14-B045 <160> 26 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> P53Bac-F <400> 1 gggagaaaac gttagggtgt gg 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> P53Bac-R <400> 2 ccaatccagg gaagcgtgtc ac 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P53P-F <400> 3 tgcaccctcc tccccaactc 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> P53P-R <400> 4 ctccctggac ggtggctcta g 21 <210> 5 <211> 213 <212> DNA <213> Artificial Sequence <220> <223> Mdsp53 promoter <220> <221> misc_difference <222> (6) <223> 5-methylcytosine <220> <221> misc_difference <222> (9) <223> 5-methylcytosine <220> <221> misc_difference <222> <223> 5-methylcytosine <220> <221> misc_difference <222> (69) <223> 5-methylcytosine <220> <221> misc_difference <222> (96) <223> 5-methylcytosine <220> <221> misc_difference <124> <223> 5-methylcytosine <220> <221> misc_difference <175> <223> 5-methylcytosine <400> 5 caggtcggcg agaatcctga ctctgcaccc tcctccccaa ctccatttcc tttgcttcct 60 ccggcaggcg gattacttgc ccttacttgt catggcgact gtccagcttt gtgccaggag 120 cctcgcaggg gttgatggga ttggggtttt cccctcccat gtgctcaaga ctggcgctaa 180 aagttttgag cttctcaaaa gtctagagcc acc 213 <210> 6 <211> 213 <212> DNA <213> Artificial Sequence <220> <223> umsp53 promoter <400> 6 caggtcggcg agaatcctga ctctgcaccc tcctccccaa ctccatttcc tttgcttcct 60 ccggcaggcg gattacttgc ccttacttgt catggcgact gtccagcttt gtgccaggag 120 cctcgcaggg gttgatggga ttggggtttt cccctcccat gtgctcaaga ctggcgctaa 180 aagttttgag cttctcaaaa gtctagagcc acc 213 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> PNA for Mdsp53 promoter <220> <221> misc_difference <222> (1) <223> NH2 linker <400> 7 aatccgcctg ccgga 15 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> DNA-1 (oligo from human GAPDH gene) (NCBI JN613429) <400> 8 tgcagcaatg cctcctgcac caccaactgc ttag 34 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> DNA-2 (oligo from elongator complex gene 3) (NCBI) <400> 9 gacaaataca cacacagcct tgccgtgaat tg 32 <210> 10 <211> 38 <212> DNA <213> Artificial Sequence <220> DNA-3 (Oligo from SP6 promoter) <400> 10 ggatgcatag cttgagtatt ctatagtgtc acctaaat 38 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 1 mCpG DNA <220> <221> misc_difference <222> (13) <223> 5-methylcytosine <400> 11 ctttcgttcc tccggcaggc ggatcgctaa 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 2 mCpG DNA <220> <221> misc_difference <222> (13) <223> 5-methylcytosine <220> <221> misc_difference <20> <223> 5-methylcytosine <400> 12 ctttcgttcc tccggcaggc ggatccgtaa 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 3 mCpG DNA <220> <221> misc_difference <222> (5) <223> 5-methylcytosine <220> <221> misc_difference <222> (13) <223> 5-methylcytosine <220> <221> misc_difference <20> <223> 5-methylcytosine <400> 13 ctttcgttcc tccggcaggc ggatccgtaa 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <222> 4 mCpG DNA <220> <221> misc_difference <222> (13) <223> 5-methylcytosine <220> <221> misc_difference <222> (5) <223> 5-methylcytosine <220> <221> misc_difference <222> (13) <223> 5-methylcytosine <220> <221> misc_difference <20> <223> 5-methylcytosine <220> <221> misc_difference (26) <223> 5-methylcytosine <400> 14 ctttcgttcc tccggcaggc ggatccgtaa 30 <210> 15 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <220> <221> misc_difference <222> (1) <223> NH2-linker <400> 15 gaggaacgaa ag 12 <210> 16 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> 1 mCpG site at -105 position <220> <221> misc_difference <222> (19) <223> 5-methylcytosine <400> 16 tccggcaggc ggattactcg cccttacttg tcatggcgac tgtccagctt tgtgccagga 60 gcctcgcagg ggttgatggg attggggttt tcccctccca tgtgctcaag actggcgcta 120 aaa 123 <210> 17 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> 1 mCpG site at -77 position <220> <221> misc_difference <47> <223> 5-methylcytosine <400> 17 tccggcaggc ggattactcg cccttacttg tcatggcgac tgtccacgtt tgtgccagga 60 gcctcgcagg ggttgatggg attggggttt tcccctccca tgtgctcaag actggcgcta 120 aaa 123 <210> 18 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> 1 site at -35 position <220> <221> misc_difference <222> (89) <223> 5-methylcytosine <400> 18 tccggcaggc ggattactcg cccttacttg tcatggcgac tgtccacgtt tgtgccagga 60 gcctcgcagg ggttgatggg attggggtcg tcccctccca tgtgctcaag actggcgcta 120 aaa 123 <210> 19 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> 1 site at +1 position <220> <221> misc_difference <216> <223> 5-methylcytosine <400> 19 tccggcaggc ggattactcg cccttacttg tcatggcgac tgtccacgtt tgtgccagga 60 gcctcgcagg ggttgatggg attggggttt tcccctccca tgtgctcaag actggcgcta 120 aaa 123 <210> 20 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <220> <221> misc_difference <222> (4) <223> C3-NH2 linker <220> <221> misc_difference &Lt; 222 > (11) <223> C3-NH2 linker <400> 20 aatccgcctg ccgga 15 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <220> <221> misc_difference <222> (6) <223> C3-NH2 linker <220> <221> misc_difference <222> (14) <223> C3-NH2 linker <400> 21 ggtcgaaaca cggtcctc 18 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PNA probe <220> <221> misc_difference <222> (6) <223> C3-NH2 linker <220> <221> misc_difference <222> (14) <223> C3-NH2 linker <400> 22 ctgagagcaa acgcaaaa 18 <210> 23 <211> 97 <212> DNA <213> Artificial Sequence <220> <223> NF-kB <220> <221> misc_difference <222> (13) <223> 5-methylcytosine <400> 23 gccaggagcc tcgcaggggt tgatgggatt ggggttttcc cctcccatgt gctcctggcg 60 ctaaaagttt tgagcttctc aaaagtctag agccacc 97 <210> 24 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> NF-kB PNA probe <220> <221> misc_difference &Lt; 222 > (13) <223> C3-NH2 linker <220> <221> misc_difference <222> (7) (8) <223> C3-NH2 linker <400> 24 gtccccaact acccta 16 <210> 25 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> AP-1 <220> <221> misc_difference <222> (18) <223> 5-methylcytosine <220> <221> misc_difference <222> (21) <223> 5-methylcytosine <400> 25 tatctacggc accaggtcgg cgagaatcct gactctgcac cctcctccct ggcgctaaaa 60 gtttggctct tctcaaaagt ctagagccac c 91 <210> 26 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> AP-1 PNA probe <220> <221> misc_difference <222> (5) (6) <223> C3-NH2 linker <220> <221> misc_difference &Lt; 222 > (13) <223> C3-NH2 linker <400> 26 cttaggactg agacg 15

Claims (32)

CpG region methylation detection of a target nucleic acid comprising the following steps:
(a) a PNA probe hybridizing to the 5 'end or the 3' end of a target nucleic acid is vertically or horizontally immobilized, or a PNA probe hybridizing to a portion of the target nucleic acid is horizontally immobilized Treating the plasmonic sensor with a target nucleic acid to form a gold nanostar-PNA probe-target nucleic acid complex;
(b) contacting MBP2 (Methyl-CpG-binding domain protein 2) protein to the complex thus formed to induce binding to the methylated CpG site of the target nucleic acid; And
(c) analyzing the light scattering spectrum according to the MBD2 protein binding of the methylated CpG site and analyzing whether the target nucleic acid CpG region is methylated through the maximum wavelength mobility (? max) analysis obtained therefrom.
2. The method of claim 1, wherein the target nucleic acid is a p53 promoter.
The method of claim 1, wherein the PNA probe or 5 "is an amine (NH ₂₎ groups are bonded at the terminal, characterized in that in the amine (NH ₂₎ group is bonded to the inner PNA probe.
4. The method of claim 3 wherein the amine (NH ₂₎ group is characterized in that coupled to the gamma position of the probe PNA backbone.
Of claim 3, wherein the 5 PNA probe if "is an amine (NH ₂₎ groups are bonded at the terminal is fixed perpendicular to the gold nano-star, if the internal PNA probe amine (NH ₂₎ group to which is bonded one PNA probe And is horizontally fixed to the gold nanostructure.
A method for determining the number of methylated CpG sites of a target nucleic acid comprising the steps of:
(a) a PNA probe hybridizing to the 5 'end or the 3' end of a target nucleic acid is vertically or horizontally immobilized, or a PNA probe hybridizing to a portion of the target nucleic acid is horizontally immobilized Treating the plasmonic sensor with a target nucleic acid to form a gold nanostar-PNA probe-target nucleic acid complex;
(b) contacting MBP2 (Methyl-CpG-binding domain protein 2) protein to the complex thus formed to induce binding to the methylated CpG site of the target nucleic acid; And
(c) measuring the light scattering spectrum due to the MBD2 protein binding of the methylated CpG site, and measuring the number of methylated CpG sites of the target nucleic acid through the maximum wavelength mobility analysis obtained therefrom.
7. The method of claim 6, wherein the target nucleic acid is a p53 promoter.
The method of claim 6, wherein said PNA probe or 5 "is an amine (NH ₂₎ groups are bonded at the terminal, characterized in that in the amine (NH ₂₎ group is bonded to the inner PNA probe.
9. The method of claim 8 wherein the amine (NH ₂₎ group is characterized in that coupled to the gamma position of the probe PNA backbone.
9. The method according to claim 8, wherein when the amine (NH ₂ ) group is bonded to the 5 'end, the PNA probe is fixed to the gold nanostructure vertically. When the amine (NH ₂ ) And is horizontally fixed to the gold nanostructure.
A method for providing information necessary for cancer diagnosis by methylation of a target nucleic acid comprising the steps of:
(a) a PNA probe hybridizing to the 5 'end or the 3' end of a target nucleic acid is vertically or horizontally immobilized, or a PNA probe hybridizing to a portion of the target nucleic acid is horizontally immobilized Treating the plasmonic sensor with a sample containing a target nucleic acid to form a gold nanostar-PNA probe-target nucleic acid complex;
(b) contacting MBP2 (Methyl-CpG-binding domain protein 2) protein to the complex thus formed to induce binding to the methylated CpG site of the target nucleic acid; And
(c) analyzing the light scattering spectrum according to the MBD2 protein binding of the methylated CpG site and analyzing whether the CpG region of the target nucleic acid is methylated through analysis of the maximum wavelength mobility (DELTA lambda max) obtained therefrom.
12. The method of claim 11, wherein the target nucleic acid is a p53 promoter.
12. The method of claim 11, wherein the PNA probe or 5 "is an amine (NH ₂₎ groups are bonded at the terminal, characterized in that in the amine (NH ₂₎ group is bonded to the inner PNA probe.
The method of claim 13 wherein the amine (NH ₂₎ group is characterized in that coupled to the gamma position of the probe PNA backbone.
14. The method according to claim 13, wherein when the amine (NH ₂ ) group is bonded to the 5 'end, the PNA probe is fixed to the gold nanostructure vertically. When the amine (NH ₂ ) And is horizontally fixed to the gold nanostructure.
A method for detecting CpG region methylation of a transcription factor binding domain in a promoter comprising the steps of:
(a) Plasmonic plasmids based on gold nanostars in which a PNA probe that hybridizes to the 5 'end or the 3' end portion of the promoter is fixed vertically or horizontally, or a PNA probe that hybridizes with a portion of the promoter is horizontally fixed Treating the sensor with a promoter to form a gold nanostar-PNA probe-promoter complex;
(b) contacting the formed complex with MBD2 (Methyl-CpG-binding domain protein 2) protein to induce binding to the methylated CpG site of the transcription factor binding domain, and then adding a transcription factor; And
(c) measuring the light scattering spectrum according to the binding of the transcription factor, and detecting whether the CpG region is methylated in the transcription factor binding domain in the promoter through analysis of the maximum wavelength mobility and the reaction rate constant obtained therefrom.
17. The method of claim 16, wherein the promoter is a p53 promoter.
17. The method of claim 16 wherein the PNA probe or 5 "is an amine (NH ₂₎ groups are bonded at the terminal, characterized in that in the amine (NH ₂₎ group is bonded to the inner PNA probe.
The method of claim 18 wherein the amine (NH ₂₎ group is characterized in that coupled to the gamma position of the probe PNA backbone.
19. The method of claim 18, wherein the 5 PNA probe if the "combined group amine (NH ₂₎ at the terminal is fixed perpendicular to the gold nano-star, if the internal PNA probe amine (NH ₂₎ group to which is bonded one PNA probe And is horizontally fixed to the gold nanostructure.

A method for measuring inhibition of transcriptional activity by CpG region methylation of a promoter comprising the steps of:
(a) Plasmonic plasmids based on gold nanostars in which a PNA probe that hybridizes to the 5 'end or the 3' end portion of the promoter is fixed vertically or horizontally, or a PNA probe that hybridizes with a portion of the promoter is horizontally fixed Treating the sensor with a promoter to form a gold nanostar-PNA probe-promoter complex;
(b) contacting the formed complex with a methylated CpG-binding domain protein 2 (MBD2) protein to induce binding to the methylated CpG site of the target nucleic acid, and then treating the RNA polymerase and the transcription factor; And
(c) assaying the activity of the promoter by measuring the light scattering spectrum due to the binding of the RNA polymerase and the transcription factor, analyzing the maximum wavelength mobility and the reaction rate constant obtained therefrom, and comparing the activity of the gene transcription Analyzing the activity.
22. The method of claim 21, wherein the promoter is a p53 promoter.
The method of claim 21, wherein the PNA probe or 5 "is an amine (NH ₂₎ groups are bonded at the terminal, characterized in that in the amine (NH ₂₎ group is bonded to the inner PNA probe.
The method of claim 23 wherein the amine (NH ₂₎ group is characterized in that coupled to the gamma position of the probe PNA backbone.
Of claim 23, wherein the 5 PNA probe if "is an amine (NH ₂₎ groups are bonded at the terminal is fixed perpendicular to the gold nano-star, if the internal PNA probe amine (NH ₂₎ group to which is bonded one PNA probe And is horizontally fixed to the gold nanostructure.
22. The method of claim 21, wherein the transcription factor is selected from the group consisting of NF-KB, YY1, c-Myc and AP-I.
A method for monitoring the competitive interaction of a transcription factor protein with methylation of a promoter CpG region comprising the steps of:
(a) Plasmonic plasmids based on gold nanostars in which a PNA probe that hybridizes to the 5 'end or the 3' end portion of the promoter is fixed vertically or horizontally, or a PNA probe that hybridizes with a portion of the promoter is horizontally fixed Treating the sensor with a promoter to form a gold nanostar-PNA probe-promoter complex;
(b) contacting the formed complex with RNA polymerase, MBD2 and a transcription factor to induce binding to the CpG region of the promoter;
(c) monitoring light scattering spectra resulting from binding of MBD2, RNA polymerase and transcription factors, and monitoring competitive interaction of transcription factor proteins through analysis of maximum wavelength mobility and rate constants obtained therefrom.
28. The method of claim 27, wherein the promoter is a p53 promoter.
The method of claim 27, wherein said PNA probe or 5 "is an amine (NH ₂₎ groups are bonded at the terminal, characterized in that in the amine (NH ₂₎ group is bonded to the inner PNA probe.
The method of claim 29 wherein the amine (NH ₂₎ group is characterized in that coupled to the gamma position of the probe PNA backbone.
30. The method of claim 29, wherein the 5 PNA probe if the "combined group amine (NH ₂₎ at the terminal is fixed perpendicular to the gold nano-star, if the internal PNA probe amine (NH ₂₎ group to which is bonded one PNA probe And is horizontally fixed to the gold nanostructure.

28. The method of claim 27, wherein the transcription factor is selected from the group consisting of NF-KB, YY1, c-Myc, and AP-I.

Images